GTP-binding proteins (G proteins) mediate critical steps in a variety of signaling and regulatory pathways. The function of G proteins in diverse biological processes depends on their ability to cycle between inactive (GDP-bound) and active (GTP-bound) conformations in a precisely regulated manner. Aberrant forms of G proteins have been implicated in the pathogenesis of variety of disease states including cancer. Consequently, G proteins and the factors that regulate their activity are potential targets for pharmacological interdiction. The experiments described in this proposal address the mechanism by which Mss4, a 13 kDa exchange factor selective for a subset of Rab family GTPases, regulates the activation of the monomeric G protein Rab3a which functions in neuronal signaling. The experimental approach will combine high resolution structural studies by X-ray crystallography with biochemical and mutational experiments in order to identify with the principal stereochemical determinants of Rab3a activation and regulation by Mss4 as well as the inactive (GDP-bound) and active (GMPPNP-bound) forms of Rab3a, the respective crystal structures will be solved and refined at high resolution. These studies will reveal the conformational changes associated with activation and provide a structural basis for the function of Mss4 and Rab3a in neuronal signaling. In parallel, mutations in Rab3a and Mss4 will be introduced to probe the binding interaction and determine the basis for the selectivity in the interaction between Mss4 and Rab family GTPases. The results, when interpreted in the context of the Mss4 and Raba3a crystal structures, will provide a clear picture of the structural and functional determinants that govern specificity and lead to the release of nucleotide. Finally, screens will be conducted for co-crystals of Mss4 bound to Rab3a or other Rav family proteins. Comparison with structural and biochemical data for other systems including EF-Tu/EF-Ts will distinguish features unique to the Mss4/Rab3a system from those that may be global determinants for nucleotide exchange. In addition, the structure of the GMPPNP-bound form of Rab3a will provide a basis for exploring the regulatory interaction between the active form of Rav proteins and specific effector proteins in the fusion/docking complex which mediates vesicle fusion with target membranes in exocytosis.